Project Summary/Abstract My laboratory's research is focused on understanding the molecular mechanisms key to the regulation of membrane protein function and to the modulation of their output signaling. We use cryo-EM and advanced classification methods, such as manifold embedding, combined with modeling and molecular dynamics to study how ligands produce shifts in conformation equilibria and bias signaling output. We are currently studying two systems that allow us to study how different types of ligands influence the gating of ion channels. We are studying the role of lipids in triggering gating of mechanosensitive channels of the MscS family, a model system of membrane tension-sensing which can yield important information about the fundamental principles governing ion channel gating and the role that lipid-protein interactions play in this process. We have a longstanding interest in the mechanism and modulation of the ryanodine receptor (RyR), a calcium release channel fundamental to heart and skeletal function. We are using manifold embedding, a machine learning-based method to analyze cryo-EM images, to better understand how small molecules and ions such as calcium and ATP affect the conformational energy landscape of the channel. We aim to shed light on the gating mechanism of this very large ion channel, a prerequisite to understanding of its modulation by protein ligands, post-translational modifications and drugs. Our approach to these fundamental problems is to use a commbination of cryo-electron microscopy, advanced image classification techniques, modelling and molecular dynamics simulations to delineate allosteric pathways mechanistically and then test proposed models with biophysical methods such as single-channel measurements, HDX-MS and mutagenesis. Our ultimate aim is to greatly increase our molecular understanding of the gating and allosteric modulation of ion channels. Progress towards such knowledge has the potential to open the way for the design of small molecule allosteric modulators with very well controlled effects on their targets, aiding the development of drugs with limited side effects. A longer-term aim is to further develp and use our tools towards gaining a better understanding of allosteric modulation in other classes of membrane proteins of therapeutic importance. In particular, G protein-coupled receptor ligands can induce the selective binding of different transducers in a process called biased signaling. The molecular mechanisms at play in this process are still poorly understood despite their fundamental importance for the development of safer drugs.